Pseudouridine is the most abundant post-transcriptionally modified nucleoside found in ribonucleic acid (RNA). Pseudouridine is an enzymatically-generated isomer of the canonical base uridine with additional hydrogen bonding capabilities, and appears to lead to local structural and biochemical effects within RNAs. Pseudouridine has been detected in several types of RNA, including ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and may be present in messenger RNA (mRNA). The prevalence of pseudouridine appears to track its biological importance. It has been implicated in a wide variety of cellular processes including gene expression, gene regulation, and steroid receptor signaling through pseudouridylation of the steroid receptor RNA activator SRA. The role of pseudouridine in gene expression is mediated through the stabilization of codon-anticodon interactions within the ribosome, read-through of stop codons through unusual base-pairing, attenuation of gene expression, stress-induced alternative splicing, rRNA stability and ribosome assembly. Recent results demonstrate that pseudouridine in mRNA can be translated by the ribosome. The current interest in determining the medical significance of post-transcriptionally modified nucleosides in RNA requires a new method that is capable of rapidly and conveniently identifying pseudouridine, quantifying this modified nucleoside, and determining its location within large RNA molecules. This proposal seeks to conduct the necessary proof-of-concept experiments to develop a mass spectrometry (MS)-based selected reaction monitoring (SRM) assay for pseudouridine that can be used to detect and localize this modified nucleoside within mammalian RNAs, including RNAs from cells in the nervous system. The proposed research plan addresses two aims: (1) Definition of technical specifications for the SRM assay including limits of detection and quantification, dynamic range, complexity of RNA sample, and accuracy of pseudouridine detection, quantification and sequence placement; and (2) Demonstration of the utility of this assay for monitoring this critical modified nucleoside in human RNAs obtained from cell lines and tissue samples. The research will also define the appropriate sample preparation steps required for turn-key analysis of RNA by the MS-based SRM assay. Success in this project will support the development of a reagent kit for use by the scientific community to investigate the clinical significance of this most prevalent RNA modification. The project will also examine the applicability of this assay to biopsy materials, which could enable its use for research into a wide range of disorders of the nervous system.